Embryonic stem cells (ESCs) maintain an epigenetic state that enables both self-renewal and differentiation into all embryonic lineages. Because of their ability to differentiate into any of the 220 cell types in adult body and their capacity for self-renewal, ES cell-based therapies have been proposed for regenerative medicine and tissue replacement after injury or disease. The development of such therapies, however, largely depends on a complete understanding of the genes essential for the self-renewal and pluripotency properties of ES cells. Focused functional as well as high throughput analysis revealed that in ES cells Oct4, Sox2, Nanog, and Klf4 form the core transcriptional circuitry to stably maintain the expression of pluripotency genes, and to repress lineage determinant genes. RNAi screens of nearly 20K genes in mouse ESCs (done by 3 different groups) collectively have revealed 400 other genes that maintain ESC cell identity. Unfortunately, although 3 these studies screened more or less the same set of genes, there is almost no agreement on which genes they report to be essential for ESC maintenance. Despite these large-scale efforts, our understanding of self-renewal still remains largely incomplete. In an effort to identify the complete set of genes essential for ESC identity, we undertook a massive effort to integrate previously published gene expression microarray datasets in mouse ESCs and differentiated cells (DCs) across various developmental stages from over 30 studies. A robust meta-analysis framework was used to analyze the expression data from each study separately to generate a ranked list of genes ordered from those that are over-expressed in ESCs to those that are under-expressed in ESCs. We performed high throughput RNAi screens of tens of genes in an effort to validate novel regulators of embryonic stem cell self-renewal and pluripotency. We have developed and applied a combination of systems, molecular, and functional genomic genomics and bioinformatics approaches to map and characterize gene regulatory networks in mouse embryonic stem cells. Our findings will help understand the role and importance of novel regulators in core stem cell transcriptional network, and how transcription regulators and epigenetic modifications regulate gene expression programs during cellular development and differentiation.